As in previous years, a major focus of this project has been detailed longitudinal study of the behavioral and biological consequences of differential early social rearing, most notably comparing rhesus monkey infants reared by their biological mothers in pens containing adult males and other mothers with same-age infants for their first 6-7 months of life (MR), with monkeys separated from their mothers at birth, hand-reared in the labs neonatal nursery for their first month and then raised in small groups of same-age peers for their next 6 months (PR). In a third standard rearing environment, surrogate-peer rearing (SPR), infants are separated from their mothers and nursery reared just like PR infants, but then at 1 month are housed in individual cages containing an inanimate surrogate mother and additionally are placed in a play cage with 3 other like-reared peers for 2 hours daily for the next are 6 months. At 7-8 months of age, MR, PR, and SPR infants are all moved into one large pen, where they all live together until puberty. Thus, the differential social rearing occurs only for the first 7-8 months; thereafter MR, PR, and SPR all share the same physical and social environment. We previously demonstrated that PR monkeys cling more, play less, tend to be more impulsive and aggressive, and exhibit much greater behavioral and biological disruption during and immediately following short-term social separation at 6 months of age than MR monkeys, and they also exhibit deficits in serotonin metabolism (as indexed by chronically low values of CSF 5-HIAA), as do SPR monkeys, and they also have significantly lower levels of 5-HTT binding throughout many brain regions than do MR subjects. Many of these differences between MR and PR monkeys persist throughout the childhood years. Research in collaboration with colleagues from NIAAA has demonstrated that both PR and SPR monkeys also consume significantly more alcohol when placed in a happy hour situation as adolescents and young adults. This past year we published data extending these rearing condition differences to include patterns of brain lateralization, cortisol concentrations in hair (a measure of chronic HPA activity), and measures of brain structure and function, as assessed by structural MRI and PET, respectively. Additional differences in measures of social dominance status, maternal competence, and physical health during childhood, adolescence, and adulthood were also documented. Somewhat surprisingly, however, MR and PR juveniles did not exhibit differential behavioral responses to chronic fluxotine treatment, and as adolescents their pattern of serotonin transporter distribution throughout their brains did not differ as a function of differential rearing but did reflect significant fluxotine treatment effects. Another major focus of recent research for this project has involved characterizing interactions between differential early social rearing and polymorphisms in several candidate genes (G X E interactions), most notably the 5HTTLPR gene. During the past year we expanded the range of outcomes for which G x E interactions involving the 5-HTTLPR polymorphism and early rearing condition differences appear, including social play, and behavioral reactions to a variety of social stressors, and in epigenetic regulation of brain activity. In addition, we recently reported significant G x E interactions between early MR vs.PR rearing and polymorphisms for several other candidate genes, including DRD1, neuropeptide Y, mu opioid (OPRMI), BDNF, NOS-1, and a SNP in the glucocorticoid gene, with outcome measures including play behavior, social buffering, behavioral and HPA reaction to an unfamiliar conspecific, naloxone treatment, alcohol consumption, and plasma BDNF concentrations. In virtually every case a similar pattern was observed: The less efficient (transcription-wise) allele was associated with a negative outcome ammong PR reared monkeys but a neutral or, in some cases, even an optimal outcome for MR reared subjects carrying that same less efficient allele, suggesting an overall buffering effect of MR rearing for individuals carrying these so-called risk alleles. Finally,this past year we published the results of two sets of studies investigating the effects of differences in early social rearing (MR vs. SPR) on genome-wide patterns of mRNA expression in leukocytes, and on methylation patterns in prefrontal cortex and in T-cell lymphocytes. Our research involving mRNA expression, carried out in collaboration with Steven Cole and James Heckman, examined expression patterns in differentially reared 4-month-old infants. In all, 521 different genes were significantly more expressed in MR infants than in SPR infants, whereas the reverse was the case for another 717 genes. In general, SPR- reared infants showed enhanced expression in genes involved in inflammation, T-lymphocyte activation and cell proliferation, and suppression of antiviral and antibacterial responses, a pattern curiously also seen in leukocyte expression in adult humans who perceive themelves as socially isolated. Since that initial study we have embarked on a prospective longitudinal study in which differentially reared subjects are being sampled at 14 days, 30 days, 6-7 months, and every 4 months thereafter until they reach puberty; preliminary results indicate that the above rearing condition diffferences in genome-wide patterns of mRNA expression in leukocytes continue to be pervasive and persistent throughout development. The other set of studies, carried out in collaboration with Moshe Szyf and his lab at McGill, involved genome-wide analyses of methylation patterns in differentially reared monkeys when they were adults. The initial study compared such patterns in prefrontal cortex tissue and T-cell lymphocytes obtained from 8-year-old monkeys differentially reared for their initial 6-7 weeks of life and thereafter maintained under identical conditions until adulthood. These analyses revealed that (a) more than 4,400 genes were differentially methylated in both PFC and lymphocytes, (b) although there was considerable tissue specificity, approximately 25% of the affected genes were identical in both PFC and lymphocytes, and (c) in both PFC and lymphocytes methylated promoters tended to cluster both by chromosomal region and gene function. We have since initiated a prospective longitudinal study of genome-wide methylation patterns in lymphocytes, collecting samples from exactly the same MR and SPR monkeys at exactly the same time points as in the afore-mentioned longitudinal study of mRNA expression. Preliminary finding suggest that, at least in lymphocites, extensive rearing conditions are present within the first month of life but can at least in part be minimized and/or re-directed subsequently following certain environmental interventions. interventions